C2, and unusual filamentous bacterial virus: protein sequence and conformation, DNA size and conformation, and nucleotide/subunit ratio.
C2, and unusual filamentous bacterial virus: protein sequence and conformation, DNA size and conformation, and nucleotide/subunit ratio.
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C2，和不常见的丝状细菌病毒：蛋白质序列和构象、DNA 大小和构象以及核苷酸/亚基比率。
DOI：
10.1021/bi00012a026
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发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
Day,LA
中科院分区：
文献类型：
--
作者：
Kostrikis,LG;Reisberg,SA;Kim,HY;Shin,S;Day,LA
材料和方法细菌生长和病毒纯化。从D. E.布拉德利（纽芬兰纪念大学，圣约翰，纽芬兰，加拿大）。S.鼠伤寒在BHI培养基（每升200克小牛脑浸液、250克牛心浸液和2.5克磷酸二钠）中生长。通过600 nm处的光散射测量和/或使用Petroff-Hauser室通过显微镜直接计数来监测细菌生长。用C2病毒感染生长至108个细菌/mL密度的细胞。感染复数约为10-20 PFU/细胞。感染的培养物在37 ℃下孵育约16小时.通过离心除去细胞，并将上清液溶液制成1 M的NaCl溶液和10%的聚乙二醇溶液，并在室温下静置2-3小时以使沉淀物形成（Yamamoto et al.，1970年）。然后通过以16000 g离心20分钟收集病毒沉淀物，并重悬于10 mM磷酸钠缓冲液（pH 7. 4）.将1 mL等分试样在相同缓冲液中的5-20%线性蔗糖梯度上分层，并在Beckman SW 41转子中以40000 g离心1.5-3 h。通过270 nm处的紫外吸收监测梯度。用10 mM磷酸钠缓冲液（pH 7.4）透析病毒级分，测定其滴度和UV吸光度。C2在CsCl和KBr中的聚集排除了使用这些盐进行浮力密度梯度纯化的可能性，因此本文所述的病毒进行了基于沉降速度的额外纯化，包括两轮沉淀差示沉降和重复蔗糖梯度分级。产量为每升感染培养物约2 mg纯化病毒。从用C2 dsDNA 1转染的大肠杆菌JM 109中获得了增加的病毒产量（Kostrikis，1993）。正如最初由布拉德利等人观察到的那样。（1982），在S.鼠伤寒沙门氏菌是可变的大小，有时很难或不可能看到。在室温下孵育平板产生最佳结果。
MATERIALS AND METHODSBacterial Growth and Virus Purification. C2 virus and strains of Salmonella typhimurium LT2 bearing IncC plasmids that serve as hosts were obtained from D. E. Bradley (Memorial University of Newfoundland, St. John’s, Newfoundland, Canada). S. typhimurium was grown in BHI medium (200 g of calf brain infusion, 250 g of beef heart infusion, and 2.5 g of disodium phosphate per liter). The bacterial growth was monitored by light-scattering measure-ments at 600 nm and/or direct counting by microscopy using a Petroff—Hauser chamber. Cells grown to a density of 108 bacteria/mL were infected with C2 virus. The multiplicities of infection were about 10-20 PFU/cell. The infected cultures were incubated at 37 C for about 16 h. The cells were removed by centrifugation, and the supernatant solution was made 1 M in NaCl and 10% in polyethylene glycol) and allowed tostand for 2—3 h at room temperature in order for the precipitate to develop (Yamamoto et al., 1970). The virus precipitate was then collected by centrifugation at16000g for 20 min and resuspended in 10 mM sodium phosphate buffer (pH7. 4). Aliquots of 1 mL were layered on 5—20% linear sucrose gradients in the same buffer and centrifuged in a Beckman SW 41 rotor at 40000g for 1.5-3 h. The gradients were drained with monitoring by ultraviolet absorption at 270 nm. The virus fractions were dialyzed against 10 mM sodium phosphate buffer (pH 7.4), and their titer and UV absorbance were determined. C2 aggregation in CsCl and KBr precluded the use of these salts for buoyant density gradient purifications, so the virus characterizedhere was subjected to additional purification based on sedimenta-tion velocity, including two rounds of pelleting differential sedimentation and a repeat sucrose gradient fractionation. Yields were approximately 2 mg of purified virus per liter of infected culture. Increased yields of virus were obtained from Escherichia coli JM109transfected with C2 dsDNA1 (Kostrikis, 1993). As originally observed by Bradley et al.(1982), we found C2 plaques on S. typhimurium to be of variable size, sometimes difficult or impossible to see. Incubation of the plates at room temperature produced the best results.